Abstract:Objective:To explore the method of isolation and biological analysis of tumor stem cell of pancreatic adenocarcinoma cell line PANC-1.
Methods :PANC-1 cells were cultured. Subpopulation cells which have properties of tumor stem cells were isolated according to the cell surface markers CD44 and CD24 by flow cytometry from pancreatic adenocarcinoma cell line PANC-1. The obtained CD44-CD24-, CD44+CD24+ and unsorted PANC-1 cells were cultured in serum-free medium (SFM). The SFM was DMEM-F12 supplemented with 10 ng/mL fibroblast growth factor, 20 ng/mL epidermal growth factor, 5 kg/mL insulin, 2.75 mg/mL transferrin, 2.75 ng/mL selenium (insulin-transferrin-selenium solution), penicillin(1×105 U/L) and streptomycin (100 mg/L). Then the proliferative capability of these cells in vitro was estimated by MTT method. The tumor growth from different subpopulation cells inoculated into nude mice was studied, and expression of CD44 and CD24 of the CD44+ CD24+ cells -formed nodules and PANC-1 cells were detected by avidin-biotin-peroxidase complex (ABC) immunohistochemical staining.
Results:Expressed the cell surface marker CD44, in 5.1% to 17.5% of sorted PANC-1 cells, 21.8% to 70.1% expressed CD24, and only 0.9% to 3.5% of cells were CD44+CD24+. Compared with CD44-CD24- cells, CD44+CD24+ cells had a lower growth rate in vitro. For the former, index growth trend appeared at 5th day, while the latter appeared at 7th day. The sorted cell population with the highest tumorigenic potential were those cells expressing CD44 and CD24.After implantation of 1×105 CD44-CD24-cells in nude mice, no tumor growth was evident at 12 weeks. In contrast, nude mice implanted with 5×103 CD44+ CD24+ cells had large tumors evident at 4 weeks(2/8), and at least a 20～50-fold increase in tumorigenic potential(P<0.05 or P<0.01). There was no obvious histological difference between the cells of the CD44+ CD24+ cells-formed nodules and PANC-1 cells.
Conclusions:CD44 and CD24 may be used as the cell surface markers for isolation of pancreatic cancer stem cells from pancreatic adenocarcinoma cell line PANC-1. Subpopulation cells CD44+CD24+ have properties of tumor stem cells.